The study of the human proteome, in particular the human serum proteome, is an area of great interest, especially with respect to the pharmaceutical industry, with its potential to identify disease or biological markers. Studying this proteome presents a major challenge due to the varying concentrations of the constituent proteins of serum. These concentrations can vary by approximately ten orders of magnitude. Most of the pharmaceutically useful proteins are of the low molecular weight type and are found in low concentrations.
Human serum is typically comprised of blood with its constituent cells (erythrocytes and leucocytes) and clotting factors removed. The protein concentration of the serum is usually in the range of from 50 to 70 mg/ml. Approximately 70% of this protein is serum albumin (30 to 35 mg/ml) and 10% is IgG (5 to 7 mg/ml).
There are at least 10,000 proteins in human serum but most, approximately 95%, are at very low concentrations and have low molecular weights. For example, interleukin 6 (a marker for inflammation and/or infection) has a molecular weight of 21 kDa and is present in serum at a concentration of 10 pg/ml; a concentration of almost ten orders of magnitude less than serum albumin.
One of the most popular methods for examining the proteome is to use two-dimensional electrophoresis (2DE). Typically, this involves the separation of proteins by their isoelectric point and then by their molecular weight by SDS-PAGE.
2DE is advantageous as it has the potential to separate several thousand proteins as spots on one gel. The spots can then be excised from the gel, digested with trypsin and identified using MALDI-MS (matrix-assisted laser desorption ionisation mass spectroscopy). Other methods used in the separation of proteins include high-performance liquid chromatography and SELDI-MS (surface-enhanced laser desorption ionisation mass spectroscopy).
However, these methods usually involve a process of prefractionation and can result in the non-specific removal of proteins of interest that are associated with other proteins that are not of interest.